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Review Article

Review on Stability Indicating Assay Methods (SIAMs)

Salma S. Quadri*, Lalit V. Sonwane, Bhagwat N. Poul, Sharada N. Kamshette
Department of Quality Assurance,
MSS’s Maharashtra College of Pharmacy, Nilanga,
Latur, Maharashtra, India.
*Salmaq13@gmail.com

ABSTRACT
The main contemporary goal of stability indicating methods is to provide information about condition
for stress testing so as to establish the stability of drug substances and product. This paper reviews the
regulatory aspects for development of stability indicating methods. SIMs are used to differentiate the
API from its potential decomposition product. Regulatory guidance in ICH Q1A (R2) ICH Q3B (R2) Q6A
and FDA 21 CFR section 211 requires validated stability indicating methods. Force degradation is
required to demonstrate the specificity when developing SIMs and for this reason, it should be perform
prior to implementing the stability studies. Force degradation of drug standard and excipients is carried
out under different conditions to determine whether the analytical method is stability indicating. The
approaches for the development of stability indicating method is discussed.

Keywords: Stability indicating method, Regulatory guidelines, Force degradation, Development of

SIAMs, Stress testing

INTRODUCTION which further helps in determination of the

Chemical stability of pharmaceutical molecules
is a matter of great concern as it affects the
safety and efficacy of the drug product. The FDA
and ICH guidance states the requirement of
stability testing data to understand how the
quality of a drug substance and drug product
changes with time under the influence of
various environmental factors. Knowledge of
the stability of molecule helps in selecting
proper formulation and package as well as
providing proper storage conditions and shelf
life, which is essential for regulatory
documentation. Forced degradation is a process
that involves degradation of drug products and
drug substances at conditions more severe than
accelerated conditions and thus generates
degradation products that can be studied to
determine the stability of the molecule. The ICH
guideline states that stress testing is intended
to identify the likely degradation products
How to cite this article: SS Quadri, LV Sonwane, B Poul, S Kamshette; Review on Stability Indicating Assay
Methods (SIAMs); PharmaTutor; 2014; 2(8); 16-31
PharmaTutor Magazine | Vol. 2, Issue 8 | magazine.pharmatutor.org
intrinsic stability of the molecule, establishing
degradation pathways and to validate the
stability indicating procedures used [1]. But
these guidelines are very general in conduct of
forced degradation and do not provide details
about the practical approach towards stress
testing. Although forced degradation studies
are a regulatory requirement and scientific
necessity during drug development, it is not
considered as a requirement for formal stability
program. It has become mandatory to perform
stability studies of new drug moiety before
filing in registration dossier. The stability studies
include long term studies (12 months) and
accelerated stability studies (6 months). But
intermediate studies (6 months) can be
performed at conditions milder than that used
in accelerated studies. So the study of
degradation products like separation,
identification and quantitation would take even

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more time. As compared to stability studies,
forced degradation studies help in generating
degradants in much shorter span of time,
mostly a few weeks. The samples generated
from forced degradation can be used to
develop stability indicating method which can
be applied latter for the analysis of samples
generated from accelerated and long term
stability studies. This review provides a proposal
on the practical performance of forced
degradation and its application for the
development of stability indicating method. The
stability-indicating assay is a method that is
employed for the analysis of stability samples in
pharmaceutical industry. With the advent of
International Conference on Harmonisation
(ICH) guidelines, the requirement of
establishment of stability-indicating assay
method (SIAM) has become more clearly
mandated. The guidelines explicitly require
conduct of forced decomposition studies under
a variety of conditions, like pH, light, oxidation,
dry heat, etc. and separation of drug from
degradation products. The method is expected
to allow analysis of individual degradation
products. A review of literature reveals a large
number of methods reported over the period of
last 3–4 decades under the nomenclature
‘stability-indicating’. However, most of the
reported methods fall short in meeting the
current regulatory requirements. Accordingly,
the purpose of this write-up is to suggest a
systematic approach for the development of
validated SIAMs that should meet the current
ICH and regulatory requirements. The
discussion also touches upon various critical
issues, such as the extent of separation of
degradation products, establishment of mass
balance, etc., which are important with respect
to the development of stability-indicating
assays, but are not yet fully resolved. Some
other aspects like suitability of pharmacopoeial
methods for the purpose and the role of SIAMs
in stability evaluation of biological/
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biotechnological substances and products are
also delved upon.
According to FDA guideline (Guidance for
Industry, Analytical Procedures and Methods
Validation, FDA, 2000), a Stability Indicating
Method (SIM) is defined as a validated
analytical procedure that accurate and precisely
measure active ingredients (drug substance or
drug product) free from process impurities,
excipients and degradation products. The FDA
recommends that all assay procedures for
stability should be stability indicating. The main
objective of a stability indicating method is to
monitor results during stability studies in order
to guarantee safety, efficacy and quality. It
represents also a powerful tool when
investigating out-of-trend (OOT) [2] or out-of-
specification (OOS) [3] results in quality control
processes.
REGULATORY STATUS OF STABILITY-
INDICATING ASSAYS
The ICH guidelines have been incorporated as
law in the EU, Japan and in the US, but in
reality, besides these other countries are also
using them. As these guidelines reflect the
current inspectional tendencies, they carry the
de facto force of regulation. The ICH guideline
Q1A on Stability Testing of New Drug
Substances and Products [4] emphasizes that the
testing of those features which are susceptible
to change during storage and are likely to
influence quality, safety and/or efficacy must be
done by validated stability-indicating testing
methods. It is also mentioned that forced
decomposition studies (stress testing) at
temperatures in 10 °C increments above the
accelerated temperatures, extremes of pH and
under oxidative and photolytic conditions
should be carried out on the drug substance so
as to establish the inherent stability
characteristics and degradation pathways to
support the suitability of the proposed
analytical procedures. The ICH guideline Q3B
entitled ‘Impurities in New Drug Products’

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emphasizes on providing documented evidence
that analytical procedures are validated and
suitable for the detection and Quantitation of
[5]
degradation products . It is also required that
analytical methods should be validated to
demonstrate that impurities unique to the new
drug substance do not interfere with or are
separated from specified and unspecified
degradation products in the drug product. The
ICH guideline Q6A, which provides note for
guidance on specification [6] also, mentions the
requirement of stability-indicating assays under
Universal Tests/Criteria for both drug
substances and drug products. The same is also
a requirement in the guideline Q5C on Stability
Testing of Biotechnological/Biological Products
[7]
. Since there is no single assay or parameter
that profiles the stability characteristics of such
products, the onus has been put on the
manufacturer to propose a stability-indicating
profile that provides assurance on detection of
changes in identity, purity and potency of the
product. Unfortunately, none of the ICH
guidelines provides an exact definition of a
stability-indicating method. Elaborate
definitions of stability-indicating methodology
are, however, provided in the United States-
Food and Drug Administration (US-FDA) stability
guideline of 1987 [8] and the draft guideline of
1998 [9]. Stability-indicating methods according
to 1987 guideline were defined as the
‘quantitative analytical methods that are based
on the characteristic structural, chemical or
biological properties of each active ingredient
of a drug product and that will distinguish each
active ingredient from its degradation products
so that the active ingredient content can be
accurately measured.’ This definition in the
draft guideline of 1998 reads as: ‘ validated
quantitative analytical methods that can detect
the changes with time in the chemical, physical,
or microbiological properties of the drug
substance and drug product, and that are
specific so that the contents of active
ingredient, degradation products, and other
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components of interest can be accurately
measured without interference.’ The major
changes brought in the new guideline are with
respect to (i) introduction of the requirement of
validation, and (ii) the requirement of analysis
of degradation products and other components,
apart from the active ingredients(s). The
requirement is also listed in World Health
Organization (WHO), European Committee for
Proprietary Medicinal Products and Canadian
Therapeutic Products Directorate’s guidelines
on stability testing of well established or
existing drug substances and products [10,11,12].
Even the United States Pharmacopoeia (USP)
has a requirement listed under ‘Stability Studies
in Manufacturing’, which says that samples of
the products should be assayed for potency by
the use of a stability-indicating assay [13]. The
requirement in such explicit manner is,
however, absent in other pharmacopoeias.
Current ICH guideline on Good Manufacturing
Practices for Active Pharmaceutical Ingredients
(Q7A), which is under adoption by WHO, also
clearly mentions that the test procedures used
in stability testing should be validated and be
stability- indicating [14].
OBJECTIVE OF STABILITY STUDIES
Stability studies are performed to establish the
shelf life and storage condition of API and
product. In recently adopted stability
guidelines, the committee for proprietary
medicinal product (CPMP) indicates the
objective of stability testing is to provide
evidence on how much quality of an API varies
with time under influence of the variety of
environmental factor such as temperature,
humidity and light. The stability of API does not
mean “fix” or “not likely change” but it means
“controlled and acceptable change”. Force
degradation condition, stress agent
concentration and time of stress are to be
establishing in such a way that, they effect
degradation preferably 10-20% of parent
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constituent. Stability testing is performed for
welfare of the patient and to protect
reputation of producer, as a requirement of
regulatory agencies to provide data that may be
of value in formulation of other product [15].
STEPS INVOLVED DURING THE DEVELOPMENT
OF STABILITY–INDICATING ANALYTICAL
METHODS (SIAMs)
A SIAMs is an estimative analytical method used
to detect a trace level amount or residual levels
of the API present due to degradation or
designing of its synthesis route. As per the FDA
regulations, a SIAMs is defined as a completely
validated method that accurately and precisely
measures API free from potential interferences
like degradants, biproducts, intermediates, and
excipients and the FDA recommend that all
assay content methodologies for stability
[16]
studies be stability indicating . There are
three components necessary for implementing
a SIAMs.
1. Generation of degraded samples for testing
selectivity of the method,
2. Method development,
3. Method validation
Step 1: Generation of degraded samples for
testing selectivity of the method
Here lies one of the main concerns related to a
development of a SIM, since the available
guidance documents do not state the extent to
which stress tests should be carried out – that
is, how much stress should be applied or how
much degradation should be aimed for. In fact,
there is not a “gold rule” that attends this issue
and therefore, it is important to keep in mind
that experimental conditions of stress tests,
should be realistic and lead to “purposeful
degradation” [17].
Stress tests should generate representative
samples to assess drug substance and drug
product stability, provide information about
possible degradation pathway and demonstrate
the stability indicating power of the analytical
procedures applied.
1) DETERMINATION OF LIMIT OF
QUANTIFICATION (LOQ)
In close relation to the determination of the
amount of degradation is the evaluation of
Limit of Detection (LOD) and Limit of
Quantification (LOQ) of the method. These
limits should be closely related to the
Reporting, Identification and Qualification of
degradation products, as stated in ICH Q3B (R2)
[5]
. These thresholds are determined either as
percentage of drug substance or total daily
intake (TDI) of degradation product. The
analytical methods are usually expected to be
validated for the ability to quantify potential
degradation products and drug impurities with
a LOD and LOQ at least as sensitive as the ICH
threshold (see Figure 1).

Figure 1: ICH thresholds for degradation products in New Drug Application (ICH Q3B)

Reporting threshold
1
Maximum daily dose Threshold2,3
≤1g 0.1%
>1g 0.05%

Identification threshold
Maximum daily dose1 Threshold 2,3
<1mg 1.0% or 5μg TDI, whichever is lower
1mg-10mg 0.5% or 20μg TDI, whichever is lower
>10mg-2g 0.2% or 2mg TDI, whichever is lower 0.10%

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Qualification threshold
1
Maximum daily dose
<10 mg
10mg – 100 mg
>100mg-2g
>2g
Note:
1. The amount of drug substance administered
per day
2. Threshold for degradation product are
express either as percentage of the drug
substance or as total daily intake (TDI) of the
degradation product. lower threshold can be
appropriate if the degradation product
unusually toxic
3. Higher threshold should be scientifically
justified.
The identification threshold (IT) varies from 0.1
to 1.0% of the labeled amount of active
ingredient in the dosage form, or from 5μg to 2
mg TDI, depending on the maximum daily
dosage in the product´s professional labeling.
The identification threshold may be lowered for
degradation products that may be exceptionally
toxic. The Reporting Threshold (RT) is either
0.1% or 0.05% depending on the maxim daily
dosage. For very low dose drug products, where
this type of sensitivity is not attainable, even
after exhaustive tentative, justification may be
provided describing the failed reports. Process-
related drug substance impurities that are also
degradation products should have the same
limits as for ICH Q3B. Ideally, the same
analytical methodology should be used for
Quality Control and Stability Studies. The
determination of Out-of-Specification or Out-of-
Trend results should be more reliable, when
using a SIM, since LOD and LOQ used allows
detection of impurities and/or degradation
products adequately. In the situation in which a
new peak arises during stability study and one
may expect that it should not exist and hence it
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Threshold 2,3
1.0% or 50μg TDI, whichever is lower
0.5% or 200μg TDI, whichever is lower
0.2% or 3mg TDI, whichever is lower
0.15%
would constitute a type of OOT, the use of a
well studied and well determined LOQ in a SIM,
will help the applicator to decide if additional
action are needed to investigate a new
substance or a OOT.
It should be mentioned that these thresholds
are established for new drug products or New
Drug Application (NDA). For Abbreviated New
Drug Application (ANDA) or generic drugs, there
are not specific regulations about this topic and
even less, the companies dealing with these
products, have background information as
those obtained in the development of NDA.
Such application is expected to contain a “full
description of the drug substance including its
physical and chemical characteristics and
stability as well; such specifications and
analytical methods are necessary to assure the
identity, strength, quality, purity and
bioavailability of drug product and stability data
with proposed expiration date”. As already
cited, for ANDA, there are not specific
regulations and the same ICH recommendation
has been used. However, precisely because of
the lack of information derived from the new
drug development, the complexity and
responsibility in developing/validating a SIM for
an ANDA is high. Information like aqueous
solubility, pH versus solubility profile, excipients
compatibility studies, etc, all information that
enable fully assume the knowledge of the
product ,will help to ensure that best (more
appropriate) condition were chosen for
developing a SIM, like those related to the
forced degradation design.
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2) Overstressing / Understressing
Care should be taken in order to avoid
overstressing or understressing samples, with
may lead to non representative or non-
purposeful degradation. So, the use of a
properly designed and executed forced
degradation study will generate representative
samples that will help to ensure that resulting
method reflects adequately long-term stability
[18]
. About the forced degradation (or stress test,
both terms will be used in the text) design, it is
[19]
recommended to include alkaline and acidic
hydrolysis, photolysis, oxidation, humidity and
temperature stress. An compilation of data
[19-23]
from literature is shown at Table 1 and
compiles the more often used conditions to
perform forced degradation studies. These
conditions can be used as a starting point in the
development of a SIM. Changing conditions to
harsher or softer levels, can be applied, when
too little or too much degradation are obtained.
For example, in cases in which too little
degradation was obtained in the hydrolyses
stress, it is recommended to increase
concentrations to 1 Mol L-1 or higher; for
oxidation stress, increase peroxide
concentration to 10% or 20% (v/v) and/or time
of reaction, as well as temperature. If co-
solvents are necessary to increase solubility, it is
recommended the use of acetonitrile that does
not work as a sensitizer in photostability stress.
Data needs to be evaluated as unusual
degradants may form with co-solvents. If even
not all conditions may cause degradation,
document efforts and severity of conditions and
should be include in final report. By the other
side, if too much degradation is detected, the
severity of conditions may be decreased, by
diluting acid/bases, neutralizing, reducing
exposure time. Also, need to be clarified, that
synthesis impurities when are not also
degradation products do not need to be
described in Stability Studies, but SIM may
assure that these impurities do not interfere on
degradation products determination.

Table 1: “More often” used conditions for forced degradation studies

Solid State

Stress Condition Period of time

Heat 60° C Up to 1 month

Humidity 75% RH Up to 1 month
Photostability 3 mm (powder) Follow ICH requirements
Exposed and non-exposed (Q1B)
samples (“control”)

Solution State

Stress Condition Period of time

Hydrolysis Acid 0.1 – 1 Mol L-1 HCl Up to weeks and 60° C
alkaline 0.1 – 1 Mol L-1 NaOH Up to weeks and 60° C
Oxidation H2O2 3% (v/v) Up to 24 hours
Photostability Exposed and non-exposed Follow ICH requirements
samples (“control”) (Q1B)

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Heat
3) FORCED DEGRADATION STUDIES (STRESS
STUDIES)
Forced degradation or stress studies are
undertaken to deliberately degrade the sample.
These studies are used to evaluate an analytical
method’s ability to measure an active
ingredient and its degradation products,
without interference, by generating potential
degradation products. During validation of the
method, drug substance are exposed to acid,
base, heat, light and oxidizing agent to produce
approximately 10% to 30% degradation of
active substance. The studies can also provide
information about the degradation pathways
and degradation products that could form
during storage. These studies may also help in
the formulation development, manufacturing,
and packaging to improve a drug product.
Reasons for carrying out forced degradation
studies include: development and validation of
stability-indicating methodology, determination
of degradation pathways of drug substances
and drug products, discernment of degradation
products in formulations that are related to
drug substances versus those that are related to
non–drug substances (e.g., excipients) [17,22].
APPROPRIATE TIMING
It is very important to know when to perform
forced degradation studies for the development
of new drug substance and new drug product.
FDA guidance states that stress testing should
be performed in Phase III of regulatory
submission process. Stress studies should be
done in different pH solutions, in the presence
of oxygen and light, and at elevated
temperatures and humidity levels to determine
the stability of the drug substance. These stress
studies are conducted on single batch. The
results should be summarized and submitted in
an annual report [24]. However, starting stress
testing early in preclinical phase or phase I of
clinical trials is highly encouraged and should be
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60° C Up to 1 month
conducted on drug substance to obtain
sufficient time for identification of degradation
products and structure elucidation as well as
optimizing the stress conditions. An early stress
study also gives timely recommendations for
making improvements in the manufacturing
process and proper selection of stability-
indicating analytical procedures [23,25].
LIMITS FOR DEGRADATION
The question of how much degradation is
sufficient has been the topic of many
discussions amongst pharmaceutical scientists.
Degradation of drug substances between 5% to
20% has been accepted as reasonable for
validation of chromatographic assays [26,27].
Some pharmaceutical scientists think 10%
degradation is optimal for use in analytical
validation for small pharmaceutical molecules
for which acceptable stability limits of 90% of
label claim is common [28]. Others suggested
that drug substance spiked with a mixture of
known degradation products can be used to
challenge the methods employed for
monitoring stability of drug product [22]. No such
limits for physiochemical changes, loss of
activity or degradation during shelf life have
been established for individual types or groups
of biological products [29].
It is not necessary that forced degradation
would result in a degradation product. The
study can be terminated if no degradation is
seen after drug substance or drug product has
been exposed to stress conditions than that
conditions mentioned in accelerated stability
protocol [30]. This is indicative of the stability of
the molecule under test. Over-stressing a
sample may lead to the formation of secondary
degradation product that would not be seen in
formal shelf life stability studies and under-
stressing may not generate sufficient
degradation products [31]. Protocols for

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generation of product-related degradation may
differ for drug substance and drug product due
to differences in matrices and concentrations. It
is recommended that maximum of 14 days for
stress testing in solution (a maximum of 24 h for
oxidative tests) to provide stressed samples for
methods development [32].
EXPERIMENTAL DESIGN
In designing forced degradation studies, it must
be remembered that more strenuous
conditions than those used for accelerated
studies (25°C/60% RH or 40°C/75% RH) should
be used. At a minimum, the following
conditions should be investigated:
(1) Acid and base hydrolysis,
(2) Hydrolysis at various ph,
(3) Thermal degradation,
(4) Photolysis, and
(5) Oxidation. For the drug substance and drug
product, the scheme shown in Figure 2 could be
used as a guide.
The initial experiments should be focused on
determining the conditions that degrade the
drug by approximately 10%. The conditions
generally employed for forced degradation are
summarized in Table 2.
However, some scientists have found it practical
to begin at extreme conditions (80°C or even
higher, 0.5N NaOH, 0.5N HCl, 3% H2O2) and
testing at shorter (2, 5, 8, and 24 hrs, etc)
multiple time points, thus allowing for a rough
evaluation of rates of degradation. Testing at
early time points may permit distinction
between primary degradants and their
secondary degradation products. This strategy
allows for better degradation pathway
determination. It must be noted that a forced
degradation study is a “living process” and
should be done along the developmental time
line as long as changes in the stability-indicating
methods, manufacturing processes, or
formulation changes are ongoing.
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Forced degradation is only considered complete
after the manufacturing process is finalized,
formulations established, and test procedures
developed and qualified. The conditions listed
in Table 1 are by no means exhaustive and
should be adjusted by the researcher as needed
to generate ~10% degradation of the API.
The nature (inherent stability/instability) of the
particular drug substance will determine in
which direction to adjust the stress conditions.
Also, the aforementioned conditions could be
used to stress the drug substance or drug
product either in the solid or liquid/suspension
form as applicable.
For oxidative degradation with H2O2, at least
one of the storage conditions should be at room
temperature. Heating H2O2 solution increases
the homolytic cleavage of the HO-OH bond to
form the alkoxy radical. The alkoxy radical is
very reactive and may come to dominate the
observed degradation pathway. Adding a small
quantity of methanol in a confirmatory stress
experiment quenches the alkoxy radical and
rules out species produced by this more
aggressive oxidizing agent. Also, the formation
of peroxycarboxymidic acid has been observed
when acetonitrile is used as a cosolvent in H2O2
stress studies (in basic conditions). The
peroxycarboximidic acid has activated
hydroxylation reactivity, which is not
representative of H2O2. To circumvent these
problems, some research scientists always
perform a parallel or alternative oxidative study
using azobisisobutyronitrile (AIBN), which is a
less reactive oxidant and has been shown to
produce more representative degradants.
List of some common conditions used in
conducting forced degradation studies for drug
substances as shown in Table 3.
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Figure 2: An Illustrative Diagram Showing the Different Forced Degradation Condition to be used for
Drug Substance and Drug Product

Table 2: Conditions Generally Employed For Forced Degradation [24]

Degradation Type Experimental Condition Storage Condition Sampling Time
Control API (no acid or base) 40 0C, 600C 1,3,5 days
0.1N NAOH 40 0C, 600C 1,3,5 days
Hydrolysis Acid Control(no API) 40 0C, 600C 1,3,5 days
Base Control(no API) 40 0C, 600C 1,3,5 days
pH: 2,4,6,8 40 0C, 600C 1,3,5 days
3% H2O2 25 0C, 600C 1,3,5 days
Peroxide control 25 0C, 600C 1,3,5 days
Oxidation Azobisisobutyronitrile (AIBN) 40 0C, 600C 1,3,5 days
AIBN Control 40 0C, 600C 1,3,5 days
Light, 1X ICH NA 1,3,5 days
Photolytic Light, 3X ICH NA 1,3,5 days
Light Control NA 1,3,5 days
Heat chamber 60 0C 1,3,5 days
Thermal Heat chamber 60 0C /75% RH 1,3,5 days
Heat chamber 80 0C 1,3,5 days
Heat chamber 80 0C /75% RH 1,3,5 days
Heat control Room Temp. 1,3,5 days

Table 3: lists some common conditions used in conducting forced degradation studies for drug
substances

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Sample condition Time / Exposure
Solid / 60 - 70ºC 7 – 10 days
Solid / 60 - 70ºC / 75% RH 10 days
Solid / simulated sunlight 2 – 3 weeks x ICH
confirmatory exposure
0.1 to 2 N HCl solutions either at RT or at 60 - 70ºC 1 – 3 days
0.1 to 1 N NaOH solutions either at RT or at 60 - 70ºC 1 – 3 days
Dilute hydrogen peroxide (0.1 to 6%) at RT or at 60 - 70ºC 1 – 3 days
Solution in Water or at 60 - 70ºC 1 – 3 days

SELECTION OF DRUG CONCENTRATION at a concentration which the drug is expected

Which concentration of drug should be used for
degradation study has not been specified in
regulatory guidance. It is recommended that
the studies should be initiated at a
concentration of 1 mg/ml [33]. By using drug
concentration of 1 mg/ml, it is usually possible
to get even minor decomposition products in
the range of detection. It is suggested that
some degradation studies should also be done
to be present in the final formulations [34]. The
reason for proposing this are the examples of
aminopenicillins and aminocephalosporins
where a range of polymeric products have been
found to be formed in commercial preparations
of containing drug in high concentrations [35].
Example-The goal is to degrade the active
moiety to 5-10% in sample for which the
conditions can be used as Shown in Table 4.

Table 4: Conditions for different pH

Study Conditions
Acidic pH 0.1N HCl
Neutral pH pH 7.0 phosphate
buffer
Basic pH 0.1N NaOH
Oxidation O2 Atmosphere, H2O2
Photolysis (UV) 1000 Watts Hrs/M2
Photolysis 6 x 106 Lux Hrs
(Fluorescence)

METHOD DEVELOPMENT AND OPTIMIZATION
Before starting method development, various
physiochemical properties like pKa value, log P,
solubility and absorption maximum of the drug
must be known for it lays a foundation for HPLC
method development. Log P and solubility helps
select mobile phase and sample solvent while
pKa value helps determine the pH of the mobile
phase [34]. Reverse phase column is a preferred
choice to start the separation of sample
components as the degradation is carried out in
aqueous solution. Methanol, water and
acetonitrile can be used as mobile phase in
various ratios for the initial stages of separation.
Selection between methanol and acetonitrile
for organic phase is based on the solubility of
the analyte. Initially the water: organic phase
ratio can be kept at 50: 50 and suitable
modifications can be made as trials proceed to
obtain a good separation of peaks. Latter buffer
can be added if it is required to obtain better
peak separation and peak symmetry. If the
method is to be extended to LC-MS then mobile
phase buffer should be MS compatible like
triflouroacetic acid and ammonium formate.
Variation in column temperature affects the

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selectivity of the method as analytes respond
differently to temperature changes. A
temperature in the range of 30-40°C is suitable
[16]
to obtain good reproducibility . It is better to
push the drug peak farther in chromatogram as
it results in separation of all degradation
products. Also a sufficient run time after the
drug peak is to be allowed to obtain the
[33]
degradants peak eluting after the drug peak .
During method development it may happen
that the drug peak may hide an impurity or
degradant peak that co-elutes with the drug.
This requires peak purity analysis which
determines the specificity of the method. Direct
analysis can be done on line by using photo
diode array (PDA) detection. PDA provides
information of the homogeneity of the spectral
peak but it is not applicable for the degradants
that have the similar UV spectrum to the drug.
Indirect method involves change in the
chromatographic conditions like mobile phase
ratio, column, etc. which will affect the peak
separation. The spectrum of altered
chromatographic condition is then compared
with the original spectra. If the degradant peaks
and area percentage of the drug peak remains
same then it can be confirmed that the drug
peak is homogeneous [36]. The degradant that
co-elutes with the drug would be acceptable if it
is not found to be formed in accelerated and
[1]
long term storage conditions . The method is
then optimized for separating closely eluting
peaks by changing flow rate, injection volume,
column type and mobile phase ratio.
METHOD VALIDATION
The developed SIM is then validated according
to USP/ICH guideline for linearity, accuracy,
precision, specificity, quantitation limit,
detection limit, ruggedness and robustness of
the method. It is required to isolate, identify
and quantitate the degradants found to be
above identification threshold (usually 0.1%)
[37,38]
. If the method does not fall within the
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26
acceptance criteria for validation, the method is
modified and revalidated [36].
Validation is defined by the International
Organization for Standardization (ISO) as
“verification, where the specified requirements
are adequate for an intended use”, where the
term verification is defined as “provision of
objective evidence that a given item fulfills
specified requirements” [39]. The applicability
and scope of an analytical method should be
defined before starting the validation process. It
includes defining the analytes, concentration
range, description of equipment and
procedures, validation level and criteria
required. The validated range is defined by
IUPAC as “the interval of analyte concentration
within which the method can be regarded as
validated” [40,41]. This range does not have to be
the highest and lowest possible levels of the
analyte that can be determined by the method.
Instead, it is defined on the basis of the
intended purpose of the method [42,43]. The
method can be validated for use as a screening
(qualitative), semi-quantitative (e.g. 5-10ppm)
or quantitative method. It can also be validated
for use on single equipment, different
equipments in the laboratory, different
laboratories or even for international use at
different climatic and environmental conditions.
The criteria of each type of validation will of
course be different with the validation level
required [39,44].
APPROACHES TO VALIDATION EXPERIMENTS
Accuracy is established by quantitation of the
sample against a reference standard for API, or
spiking placebo with API for drug product. It can
also be determined by comparison of results
from alternate measurement techniques.
Precision is determined by multiple
measurements on an authentic, homogeneous
set of samples. Samples may be analyzed on
different days, by different analysts, on
different instruments, or in different
laboratories. There are three levels of precision

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validation evaluations – repeatability,
intermediate precision, and reproducibility.
Repeatability is a measure of precision under
the same conditions over a short period of time.
Intermediate precision is a measure of precision
within the same laboratory by different
operators, using different instruments, and
making measurements on different days.
Reproducibility assesses precision between two
or more laboratories.
Specificity can be established by a number of
approaches, depending on the intended
purpose of the method. The ability of the
method to assess the analyte of interest in a
drug product is determined by a check for
interference by placebo. Specificity can be
assessed by measurement of the API in samples
that are spiked with impurities or degradants, if
available. If API-related compounds are not
available, drug can be stressed or force-
degraded in order to produce degradation
products. In chromatographic separations,
apparent separation of degradants may be
confirmed by peak purity determinations by
photodiode array, mass purity determinations
by mass spectroscopy (MS), or by confirming
separation efficiency using alternate column
chemistry. During forced degradation
experiments, degradation is targeted at 5 to
20% degradation of the API, in order to avoid
concerns about secondary degradation.
The limit of Detection and limit of Quantitation
are based on measurement Signal-to-noise
ratios of 3 and 10, respectively. Standards or
samples at concentrations near the expected
limits are measured. Signal- to-noise can be
generated by software, manually measured,
estimated from standard deviation calculations,
or limits may be empirically determined.
Linearity is established by measuring response
at various concentrations by a regression plot,
typically by method of least squares. The
response may require mathematical
manipulation prior to linearity assessments. A
visual inspection of the linearity plot is the best
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27
tool for examining proportionality of the
response. The range is established by the
required limits of the method and the point at
which linearity is compromised.
Robustness is typically assessed by the effect
of small deliberate changes to chromatographic
methods on system suitability parameters such
as peak retention, resolution, and efficiency.
Experimental factors that are typically varied
during method robustness evaluations include
[45]
:
(i) Age of standards and sample preparations,
(ii) Sample extraction time,
(iii) Variations to pH of mobile phase,
(iv) Variation in mobile phase composition,
(v) Analysis temperature,
(vi) Flow rate,
(vii) Column lot and/or manufacturer,
(viii) Type and use of filter against
centrifugation. Robustness experiments are an
ideal opportunity to utilize statistical design of
experiments, providing data driven method
control.
The ICH guidance on validation separates types
of methods according to the purpose of the
method and lists which evaluations are
appropriate for each type [28].
The ICH guidance also suggests detailed
validation schemes relative to the intended
purpose of the methods. It lists recommended
data to report for each validation parameter.
Acceptance criteria for validation elements
must be based on the historical performance of
the method, the product specifications, and
must be appropriate for the phase of drug
development.
OTHER ANALYTICAL METHODS FOR
DEVELOPING SIM
Stability-indicating methods will be
characterized by potency, purity and biological
activity [46]. The selection of tests is product
specific. Stability indicating methods may
include various methods like electrophoresis

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(SDS-PAGE, immunoelectrophoresis, Western
blot, isoelectrofoccusing), high-resolution
chromatography (e.g., reversed phase
chromatography, SEC, gel filtration, ion
exchange, and affinity chromatography) and
[47]
peptide mapping . The analytical method of
choice should be sensitive enough to detect
impurities at low levels (i.e., 0.05% of the
analyte of interest or lower) and the peak
responses should fall within the range of
detector's linearity. The analytical method
should be capable of capturing all the impurities
formed during a formal stability study at or
below ICH threshold limits [48,49]. Degradation
product identification and characterization are
to be performed based on formal stability
results in accordance with ICH requirements.
Conventional methods (e.g., column
chromatography) or hyphenated techniques
(e.g., LC–MS, LC–NMR) can be used in the
identification and characterization of the
degradation products. Use of these techniques
can provide better insight into the structure of
the impurities that could add to the knowledge
space of potential structural alerts for
genotoxicity and the control of such impurities
with tighter limits [37,47,50]. It should be noted
that structural characterization of degradation
products is necessary for those impurities
formed during formal shelf-life stability studies
[48]
and above the qualification threshold limit .
New analytical technologies that are
continuously being developed can also be used
when it is appropriate to develop stability
indicating method [51]. The unknown impurity,
which is observed during the analysis,
pharmaceutical development, stress studies
and formal stability studies of the drug
substances and drug product, can be separated
and analyzed by using various chromatographic
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28
techniques like Reversed Phase High
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Thin Layer Chromatography (TLC), Gas
Chromatography (GC), Capillary Electrophoresis
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the isolation of degradants and impurities [50].
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Stability-indicating method is an analytical
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Forced degradation studies are indispensable in
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validation protocol. Forced degradation studies
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degradation products the use of properly
designed and executed forced degradation
study will generate a representative sample
that will in turn help to develop stability-
indicating method.
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